52 HYALURONIC ACID IMPROVES CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 138
Author(s):  
L. Boccia ◽  
M. Rubessa ◽  
M. De Blasi ◽  
S. Di Francesco ◽  
G. Albero ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
In Vitro Culture ◽  
Developmental Stage ◽  
Production Efficiency ◽  
Embryo Quality ◽  
Survival Rates ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Morphological Criteria

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine fluids, has been shown to successfully support in vitro development of bovine embryos (Stojkovic et al. 2002 Reproduction 124, 141–153). The aim of this study was to evaluate the influence of high concentrations of hyaluronic acid (HA) during late in vitro culture on blastocyst development, as well as on their cryotolerance after cryotop vitrification in buffalos. In vitro matured and fertilized buffalo oocytes (n = 1007) from slaughterhouse ovaries were cultured for 4 days in SOFaa supplemented by 8 mg mL–1 of BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2 and 88% N2, in humidified air, at 38.5°C. On Day 4, cleavage rate was assessed (75.2%) and all of the cleaved elements were divided into 3 different late culture groups: 8 mg mL–1 of BSA (n = 244; group A), 8 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 251; group B) and 1 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 262; group C). On Day 7 after IVF, embryo outcome was assessed and all of the embryos were vitrified by cryotop [De Rosa et al. 2007 Ital. J. Anim. Sci. 6 (Suppl 2), 747–750] and cultured for 24 h. The resistance to cryopreservation was evaluated by assessing the survival rate on the basis of morphological criteria and the percentage of embryos reaching a more advanced developmental stage after 24 h culture. Data were analysed by the chi-square test. No differences in blastocyst rate were recorded among groups (43.9, 44.3 and 40.0%, respectively in A, B and C groups). However, out of the total embryos, a higher percentage of Grade 1 hatched blastocysts (Robertson and Nelson 1998 Manual of the International Embryo Transfer Society 9, 103–16) was observed in group C (P < 0.05) than in groups A and B (14.3, 18.8 and 25.5% in A, B and C groups, respectively). Although the supplementation with HA did not improve the survival rates following vitrification-warming (51.1, 59.4 and 58.4% in A, B and C groups, respectively), the percentage of vitrified-warmed embryos that resumed development and reached a more advanced developmental stage after culture increased (P < 0.01) in group C (20.7, 27.7 and 37.6% in A, B and C groups, respectively). In conclusion, the addition of 6 mg mL–1 of HA, together with a limited protein source (i.e. 1 mg mL–1 of BSA), during late culture improved buffalo embryo quality, indicated by both the greater percentage of advanced-stage embryos and by the resumption of development after post-warming culture.

133 L-CARNITINE DURING IN VITRO CULTURE ENHANCES THE CRYOTOLERANCE OF BUFFALO (BUBALUS BUBALIS) IN VITRO-DERIVED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 214 ◽  
Author(s):  
L. Boccia ◽  
M. De Blasi ◽  
G. Zullo ◽  
V. Longobardi ◽  
D. Vecchio ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Survival Rates ◽  
Bubalus Bubalis ◽  
Carnitine Supplementation ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Chi Square ◽  
Morphological Criteria

In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237–256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632–1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25 mM for the experiment. Cumulus–oocytes complexes (n = 288, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). On Day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg mL–1 BSA, in the absence (control, n = 143) or presence of 0.25 mM L-carnitine (n = 145). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5 M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747–750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24 h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24 h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P < 0.01). In conclusion, these results demonstrated that L-carnitine supplementation of culture medium improves the resistance to cryopreservation of in vitro produced buffalo embryos. We speculate that the increased cryotolerance observed in the presence of L-carnitine may be due to a better utilization of the endogenous lipid stores, resulting in improved embryo quality.

135 EFFECT OF L-ERGOTHIONEINE SUPPLEMENTATION DURING CULTURE ON IN VITRO EMBRYO DEVELOPMENT IN BUFFALO (BUBALUS BUBALIS)

2015 ◽  
Vol 27 (1) ◽  
pp. 160
Author(s):  
G. Zullo ◽  
A. Salzano ◽  
G. Bifulco ◽  
V. Longobardi ◽  
G. Albero ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Production Efficiency ◽  
Cell Function ◽  
Embryo Viability ◽  
Cleavage Rate ◽  
Mammalian Embryo ◽  
Morphological Criteria

It is known that in vitro mammalian embryo development is negatively affected by the increased oxidative stress occurring under culture conditions. The oxidative damage of cell components via reactive oxygen species interferes with proper cell function. Buffalo embryos are particularly sensitive to oxidative stress because of their high lipid content (Boni et al. 1992 Acta Med. Vet. 38, 153–161). l-Ergothioneine (LE) is a powerful scavenger of hydroxyl radicals (OH) and an inhibitor of iron or copper ion-dependent generation of OH from hydrogen peroxide (H2O2). The aim of this study was to evaluate whether enriching the in vitro-culture medium with LE improves in vitro embryo production efficiency in buffalo. Abattoir-derived buffalo oocytes (n = 854, over 6 replicates) were in vitro matured and fertilized according to standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). Twenty hours after IVF presumptive zygotes were cultured in SOFaa supplemented by 8 mg mL–1 BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2, 88% N2, in humidified air, at 38.5°C with 0 (control; n = 214), 0.05 mM LE (n = 217), 0.1 mM LE (n = 204), and 1 mM LE (n = 219). Cleavage rate was assessed at the time of change of culture (Day 5) and the cleaved elements were cultured for a further 2 days. The embryos obtained by the end of culture, i.e. on Day 7 post-IVF, were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson, Nelson 2010 Manual of the International Embryo Transfer Society 86–105). The percentages of total transferable embryos and Grade 1 and 2 blastocysts in relation to cleaved oocytes were recorded. Because the chronology of development is known to be one of the most reliable parameters for assessing quality, the percentage of fast-developing embryos, i.e. hatched and expanded blastocysts, was also recorded. Data were analysed by Chi-squared test. Cleavage rate was not affected by the treatment (71.4, 66.8, 68.7, and 63.0%, respectively, with 0, 0.05, 0.1, and 1 mM LE). The total embryo output increased in groups supplemented with 0.05 and 0.1 mM LE (31.3, 42.2, 43.8, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). However, the enrichment of in vitro culture with 0.1 mM LE also increased the percentage of Grade 1 and 2 blastocysts compared with the control and to 1 mM LE (21.6, 30.9, 33.9, and 21.7%, respectively, with 0, 0.05, 0.1, and 1 mM LE; P < 0.05). Likewise, 0.1 mM LE gave higher percentages of fast developing embryos than the control and 1 mM LE groups. In conclusion, these results demonstrated a beneficial effect of LE during culture on buffalo in vitro embryo development. The dose response trial indicated that the optimal concentration is 0.1 mM that also influenced the chronology of development and hence embryo viability.

1 AUTOCRINE COMMUNICATION BETWEEN BOVINE EMBRYOS CULTURED IN Primo Vision DISHES® OUTWEIGHS POSSIBLE NEGATIVE INFLUENCES OF BAD EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 115
Author(s):  
E. Wydooghe ◽  
L. Vandaele ◽  
A. Van Soom
Keyword(s):  
Developmental Stage ◽  
Odds Ratio ◽  
Fixed Effects ◽  
Binary Logistic Regression Model ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Group Culture ◽  
Individual Culture

Group culture is being used extensively for mammalian embryos, but has not been adopted so far in human embryo culture. Some doubts about its possible benefits remain because it has been hypothesised that bad quality embryos might have a negative effect on other embryos. New group culture devices have been designed allowing individual follow-up of embryos, such as Primo Vision dishes® (well of-the-well for 10 embryos in group culture; Cryo-Innovation, Budapest, Hungary). By using Primo Vision dishes®, we investigated the influence of the developmental stage of the neighbours and co-cultured embryos on the outcome at 192 h post-insemination (hpi) of a particular embryo compared with its individually cultured counterparts. Bovine presumed zygotes (n = 789; 4 replicates) produced in vitro were randomly allocated to Primo Vision dishes® or individual culture in SOF supplemented with 0.4% BSA and insulin, transferrin, selenium (Wydooghe et al. 2013 Reprod. Fertil. Dev. Epub). Cleavage rate was checked at 45 hpi: 5- to 8-cell embryos were classified as fast embryos and 2- to 4-cell embryos as slow embryos. Blastocyst development was evaluated at 192 hpi. Moreover, to evaluate which embryos benefited most from being in group (fast or slow embryos), we looked retrospectively at the influence of the developmental stage of the neighbours and the co-cultured embryos on blastocyst development. This was done separately for slow and fast embryos compared with their individually cultured counterparts. Statistical analysis was done using a binary logistic regression model, with group and replicate as fixed effects. Blastocyst development in Primo Vision dishes® was significantly better than individual culture (39.0% v. 28.5%). This beneficial outcome was mainly caused by a higher blastocyst development of slow embryos. A markedly higher percentage of slow embryos developed into a blastocyst at 192 hpi if they were surrounded by many embryos that also developed into a blastocyst, compared with individually cultured slow embryos (odds ratio: 3.0). In this study, we showed that embryos that were not cleaved at 45 hpi did not negatively affect the potential of their neighbours to become a blastocyst at 192 hpi, regardless of whether the embryo in question was a fast or a slow embryo. However, when fast embryos were in a less than favourable environment, meaning that less than 30% of their co-cultured embryos reached the blastocyst stage, blastocyst development was compromised compared with individual culture of fast embryos (odds ratio: 0.3). From our results, we clearly show that Primo Vision dishes® can combine the benefits of group culture (autocrine communication) and individual culture (individual follow-up). Taking fast embryos out of the Primo Vision dish® for further individual culture while slow embryos remain in group is a possible approach to increase total blastocyst rates.

Cryotolerance of in vitro-produced porcine blastocysts is improved when using glucose instead of pyruvate and lactate during the first 2 days of embryo culture

2013 ◽  
Vol 25 (5) ◽  
pp. 737 ◽  
Author(s):  
M. Castillo-Martín ◽  
M. Yeste ◽  
R. Morató ◽  
T. Mogas ◽  
S. Bonet
Keyword(s):  
Survival Rate ◽  
Treatment Group ◽  
In Vitro Culture ◽  
Embryo Development ◽  
Embryo Culture ◽  
Embryo Quality ◽  
Cleavage Rate ◽  
Apoptosis Index ◽  
Number Of Cells

The objective of the present study was to determine the effects of replacing glucose with pyruvate and lactate during the first 48 h of in vitro culture (IVC) in NCSU-23 medium on embryo development, embryo quality and survival of porcine blastocysts after vitrification. To this end, in vitro-produced (IVP) porcine oocytes were cultured with either glucose for 6 days (IVC-Glu) or pyruvate–lactate from Day 0 to Day 2 and then with glucose until Day 6 (IVC-PyrLac). Blastocysts were vitrified on Day 6 using the Cryotop device and, after warming, survival rate and the apoptosis index were evaluated after 24 h incubation in NCSU-23 medium. No significant differences were observed between IVC-Glu and IVC-PyrLac in terms of cleavage rate, blastocyst yield, total number of cells per blastocyst or the apoptosis index (1.82 ± 0.75% vs 3.18 ± 0.88%, respectively) of non-vitrified embryos. However, a significant increase was seen in hatching/hatched blastocysts in the IVC-PyrLac compared with IVC-Glu treatment group (12.71 ± 1.20% vs 3.54 ± 0.47%, respectively). Regardless of treatment, vitrification impaired the survival rate and the apoptosis index. When comparing both treatments after warming, the percentage of apoptotic cells was significantly higher for blastocysts in the IVC-PyrLac compared with IVC-Glu group (18.55 ± 3.49% vs 9.12 ± 2.17%, respectively). In conclusion, under the conditions of the present study, replacement of glucose with pyruvate–lactate during the first 48 h of culture resulted in a lower cryotolerance of IVP porcine embryos.

26 Season affects cryotolerance of in vitro-produced buffalo embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 139 ◽  
Author(s):  
M. A. Kosior ◽  
E. Parente ◽  
F. Salerno ◽  
K. Annes ◽  
R. Annunziata ◽  
...  
Keyword(s):  
Developmental Stage ◽  
Breeding Season ◽  
Embryo Quality ◽  
Sucrose Solution ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Seasonal Effect ◽  
Morphological Criteria ◽  
Breeding Seasons

Buffaloes are tendentially short-day breeders, and seasonality is one of the main factors affecting the feasibility of ovum pickup and in vitro embryo production technology in this species. An improvement of oocyte developmental competence during decreasing daylight months was previously reported in Italian Mediterranean buffalo (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The aim of this work was to evaluate whether season also affects embryo quality and cryotolerance. Abattoir-derived buffalo cumulus-oocyte complexes were collected during the breeding season, characterised by decreasing daylight length (n=349 over 6 replicates), and the non-breeding season, characterised by increasing daylight length (n=770 over 12 replicates). Buffalo cumulus-oocyte complexes were in vitro matured, fertilized, and cultured according to standard procedures (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality and developmental stage, and the percentages of total transferable embryos (tight morulae and blastocysts) were recorded. Embryos (n=107 and 110 in the breeding and non-breeding seasons, respectively) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5M sucrose (Boccia et al. 2013 Ital. J. Anim. Sci. 12, 492-496). Warming was carried out by plunging the cryotop strip into a 0.25M sucrose solution and transferring the embryos into 0.15M sucrose for 5min. Embryos were then washed and cultured in SOF for 24h to evaluate post-culture viability. The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and development rate (i.e. the percentage of embryos that resumed their development and reached a more advanced developmental stage) after 24h post-warming culture. Data were analysed by Student’s t-test. Both cleavage (82.8±4.3v. 73.1±1.7 in the breeding and non-breeding seasons, respectively; P&lt;0.05) and blastocyst (32.9±3.5v. 18.3±1.7 in the breeding and non-breeding seasons, respectively; P&lt;0.01) rates increased during the breeding season, confirming previous observations. Due to the different efficiency, a higher number of replicates was required during the non-breeding season to obtain an equal number of embryos. In addition, a seasonal effect was recorded on embryo quality, indicated by poorer cryotolerance of in vitro-produced buffalo embryos during the non-breeding season. Indeed, both survival (94.6±2.7% and 74.0±5.5% in the breeding and non-breeding seasons, respectively; P&lt;0.01) and development (67.3±7.6% and 40.0±7.2% in the breeding and non-breeding seasons, respectively; P&lt;0.01) rates of vitrified blastocysts decreased after 24h post-warming culture in the non-breeding season. These findings suggest that the reduced developmental competence of buffalo oocytes during the non-breeding season may also lead to lower blastocyst quality. This is in contrast to the evidence in cattle that embryo quality is mainly determined by culture conditions, whereas blastocyst production depends on oocyte quality.

88 ENRICHMENT OF CULTURE MEDIUM WITH CROCETIN IMPROVES IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2016 ◽  
Vol 28 (2) ◽  
pp. 173
Author(s):  
G. Zullo ◽  
J. E. Tamayo Palacio ◽  
C. De Canditiis ◽  
V. Longobardi ◽  
A. Salzano ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Production Efficiency ◽  
Culture Medium ◽  
Culture Conditions ◽  
Active Components ◽  
Cleavage Rate ◽  
Mammalian Embryo ◽  
Morphological Criteria

The high incidence of developmental failure of bovine in vitro-produced embryos is due to suboptimal culture conditions that induce oxidative stress. Indeed, increased oxidative stress is one of the main factors affecting in vitro mammalian embryo development, decreasing the viability of IVP embryos. It is known that saffron has a powerful antioxidant capacity, mainly due to its active components crocin and crocetin. The aim of this study was to evaluate whether enriching the in vitro culture medium with crocetin improves in vitro embryo production efficiency in cattle. The range of concentrations of crocetin was chosen after a preliminary dose response trial (322 total presumptive zygotes were cultured with 0, 1, 10, and 50 μM, over 2 replicates) that showed beneficial and deleterious effects, respectively, with the lowest and highest concentration compared with the control (36.6 ± 5.6, 57.4 ± 4.5, 46.4 ± 4.4, and 6.8 ± 3.7% blastocyst rates, respectively, with 0, 1, 10, and 50 μM; P < 0.01). Therefore, the range of concentrations to test was reduced. Abattoir-derived bovine oocytes (n = 832, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium with 0 (control; n = 208), 1 μM (n = 208), 2.5 μM (n = 208), and 5 μM (n = 208), at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality, based on morphological criteria, and for developmental stage, as previously described (Robertson and Nelson 2010, Manual of the IETS, 86–105). The percentages of total transferable embryos and grade 1 and 2 blastocysts were recorded. As the chronology of development is a reliable parameter to assess quality, the percentage of fast-developing embryos (i.e. hatched and expanded blastocysts) was also compared among groups. Differences among groups were analysed by ANOVA, and Tukey method was used as a post-hoc test. Data are presented as means ± s.d. The supplementation of crocetin during culture did not affect cleavage rate (74.9 ± 6.3, 76.4 ± 8.4, 81.4 ± 4.3, and 76.4 ± 8.4%, respectively, with 0, 1, 2.5, and 5 μM). However, post-fertilization embryo development improved with 1 µM crocetin compared with the control, both in terms of total embryo output (43.8 ± 4.4, 61.1 ± 5.2, 50.4 ± 6.7, and 53.3 ± 7.3%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.01) and grade 1 and 2 blastocysts (41.0 ± 3.6, 54.3 ± 5.4, 46.2 ± 6.7, and 49.4 ± 6.5%, respectively, with 0, 1, 2.5, and 5 μM; P < 0.05), whereas no differences were observed among the other groups. Moreover, the percentage of fast developing embryos increased with 1 µM (P < 0.05) crocetin compared with the control, with no other differences recorded among groups (17.7 ± 5.8, 34.7 ± 5.7, 24.9 ± 5.1, and 28.7 ± 7.8%, respectively, with 0, 1, 2.5, and 5 μM). In conclusion, these results demonstrated a beneficial effect of low concentrations of crocetin (1 μM) during culture both on blastocyst yield and quality, as indicated by the improved chronology of embryo development.

scholarly journals Increased Fertilization Rates after In Vitro Culture of Frozen-Thawed Testicular Immotile Sperm in Nonobstructive Azoospermic Patients

ISRN Urology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
R. Nuñez-Calonge ◽  
S. Cortes ◽  
M. Gago ◽  
P. López ◽  
P. Caballero-Peregrin
Keyword(s):  
In Vitro Culture ◽  
Embryo Quality ◽  
Clinical Pregnancy ◽  
Nonobstructive Azoospermia ◽  
Cleavage Rate ◽  
Testicular Spermatozoa ◽  
Fertilization Rates ◽  
Group I ◽  
Group Ii

Objective. To optimise the use of freeze/thaw testicular immotile spermatozoa from nonobstructive azoospermia patients and to analyse the outcome of intracytoplasmic sperm injection (ICSI) of such spermatozoa. Methods. Testicular specimens were retrieved and cryopreserved from forty patients with nonobstructive azoospermia and underwent one cycle with thawed spermatozoa (Group I) that led to pregnancy in sixteen cases. Twenty-four patients of group I underwent treatment with the same batch of thawed spermatozoa (Group II). For the first ICSI attempt, injection was performed when motile spermatozoa were found. In group II, injection was performed when maximum motility was reached. We compared mean of fertilization rate, embryo quality, clinical pregnancy rate and embryo implantation rate. Results. The mean percentage of motility was significantly higher in the group II than in the group I (18, 6 versus 8, 2). Group I showed a significant decrease in fertilization rates when compared with cryopreserved testicular spermatozoa in group II (54% versus 72%, ). No difference was noted between the cleavage rate, embryo quality, clinical pregnancy rates and implantation rates among group II and I. Conclusion. Fecundation rate can be significantly improved after in-vitro culture and sperm selection of frozen-thawed immotile testicular spermatozoa in patients with nonobstructive azoospermia.

132 RESVERATROL DURING IN VITRO CULTURE IMPROVES CRYOTOLERANCE OF IN VITRO PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 213 ◽  
Author(s):  
A. M. Abdel-Wahab ◽  
G. Zullo ◽  
L. Boccia ◽  
M. De Blasi ◽  
V. Longobardi ◽  
...  
Keyword(s):  
Standard Procedure ◽  
Survival Rates ◽  
Cancer Chemoprevention ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Positive Effects ◽  
Morphological Criteria

Despite the great improvement of in vitro embryo production (IVEP) efficiency recorded over the years in cattle, the in vitro produced (IVP) embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. One of the major factor impairing in vitro embryo development is oxidative stress. Resveratrol is an important antioxidant polyphenolic compound found in several vegetal sources, that contributes to red wine’s beneficial effects on the prevention of human cardiovascular disease. Recently, the interest in resveratrol has increased exponentially following the major findings that this molecule has positive effects on cancer chemoprevention, cardioprotection, inflammatory processes, several aspects of metabolism, leading to increased lifespan of various organisms from yeasts to vertebrates (Pirola et al. 2008 IUBMB Life 60, 323–332). A positive effect of resveratrol on in vitro embryonic development was demonstrated in swine (Lee et al. 2010 J. Reprod. Dev. 56, 330–335). The aim of this study was to evaluate whether supplementation of culture medium with resveratrol improves in vitro blastocyst development and the embryo resistance to cryopreservation in cattle. A preliminary dose response trial indicated that the optimal concentration in the range tested (from 0.5 to 10 µM) was 0.5 µM, with evident toxic effects at concentration higher than 5 µM. Abattoir-derived oocytes (n = 581, over 5 replicates) were matured and fertilized in vitro according to our standard procedure (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium, supplemented with 5% bovine serum, in the absence (control, n = 271) or presence of 0.5 µM resveratrol (n = 310) at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7 (IVF = Day 0), embryo yields were assessed and the blastocysts (except the hatched blastocysts) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (Rubessa et al. 2011). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and hatching rate after 48 h culture. Data were analyzed by chi-square test. Resveratrol supplementation during culture did not affect either cleavage (69.1 v. 72.0%, in the control and resveratrol groups, respectively) or blastocyst yields (38.3 v. 36.3%, in the control and resveratrol groups, respectively). However, treatment with resveratrol increased the cryotolerance of IVP embryos, as indicated by higher survival rates (74.7 v. 88.4%, in the control and resveratrol groups, respectively; P < 0.05) and hatching rates (35.1 v. 53.8%, in the control and resveratrol groups, respectively; P = 0.06) at 48 h. In conclusion, these results demonstrated that resveratrol supplementation during culture improves the quality, and hence the resistance to cryopreservation, of IVP bovine embryos.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

137 EFFECT OF POST-IVF DEVELOPMENTAL KINETICS ON SURVIVAL AND QUALITY OF VITRIFIED - WARMED DOMESTIC CAT BLASTOCYSTS

2007 ◽  
Vol 19 (1) ◽  
pp. 186
Author(s):  
T. Tsujioka ◽  
C. Otzdorff ◽  
J. Braun ◽  
S. Hochi
Keyword(s):  
Survival Rates ◽  
Cell Number ◽  
Domestic Cat ◽  
Minimum Volume ◽  
Cleavage Rate ◽  
Exact Probability ◽  
Cell Staining ◽  
Number Of Cells ◽  
Cooling Procedure

A limited number of reports are available for cryopreservation of IVF-derived cat blastocysts (Karja et al. 2006 Theriogenology 65, 415–423). In the present study, the IVF-derived domestic cat blastocysts exhibiting differences in their developmental kinetics were cryopreserved by vitrification. Pre- and post-warm blastocysts were examined for their cell number, and the survival rate was determined by in vitro culture of the post-warm embryos. Cumulus–oocyte complexes, recovered from the ovaries of post-pubertal queens, were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Oocytes were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. Newly formed blastocysts were harvested 6 and 7 days post-IVF and subjected to vitrification (Hochi et al. 2004 Theriogenology 61, 267–275) by a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice. The post-warm blastocysts were cultured for 24 h, and complete re-expansion was considered to be an indicator of survival. Embryos were differentially stained with Hoechst 33342 and propidium iodide to assess the total number of cells and the ICM ratio in the blastocysts. The cleavage rate of oocytes was 47.1% (314/666) and the percentage of oocytes developing to blastocysts on Day 6 and Day 7 was 9.8 and 9.5%, respectively (total blastocyst yield: 19.2%; 128/666). Post-warm in vitro survival rates of blastocysts harvested at Days 6 and 7 were 73.8% (31/42) and 66.7% (18/27), respectively (P = 0.59; exact probability test). There were no significant differences in the total number of cells and the ICM ratio between fresh control and vitrified blastocysts (P ≥ 0.05; ANOVA), although the ICM ratio of surviving Day 7 blastocysts was significantly smaller than that of fresh controls (18.9 vs. 28.9%; P = 0.03), as shown in Table 1. These results indicate that IVF-derived domestic cat blastocysts can survive vitrification by a minimum-volume cooling procedure without a reduction in the parameters studied, as long as the blastocysts are harvested on Day 6. Table 1. Differential cell staining of fresh and vitrified Day 6 and Day 7 cat blastocysts

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